The Huatanay River in Cusco-Peru, is affected by wastewater discharges along its course. In order to evaluate this impact, we evaluate antibiotic residues and their impact on the increase of bacterial resistance in the city of Cusco treatment plant. For this purpose, water samples from the influent and effluent of the treatment plant were analyzed by chromatographic methods; additionally, sensitivity tests were performed with three bacterial strains (Escherichia coli, Salmonella sp., and Klebsiella sp.), which were isolated from the same place. Six antibiotic residues were identified (ceftriaxone, amoxicillin, trimethoprim, sulfamethoxazole, dicloxacillin, and lincomycin). Those found in the highest concentration were: amoxicillin (91495 and 0 µg/L) and lincomycin (33970 and 10800 µg/L) in the influent and effluent, respectively. There is more resistance in the effluent than the influent in the case of E. coli shows resistance in the effluent to cephalexin (30 µg) and azithromycin (15 µg). Salmonella sp. is resistant to amoxicillin (15 µg), dicloxacillin (1 µg), lincomycin (2 µg), ceftriaxone (30 µg), cephalexin (30 µg), and ciprofloxacin (5 µg). Finally, Klebsiella sp. is sensitive to ceftriaxone (30 µg), amoxicillin (15 µg), and cephalexin (30 µg). This confirms that the antibiotic residues contained in the wastewater of Cusco generate resistance in the isolated bacteria.